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• W2050161362 abstract "Gene-targeted mice have recently revealed a role for lymphocytes and interferon-γ (IFNγ) in conferring protection against cancer, but the mechanisms remain unclear. Here, we have characterized a successful primary antitumor immune response initiated by naive CD4+ T cells. Major histocompatibility complex class II (MHC-II)-negative myeloma cells injected subcutaneously into syngeneic mice were surrounded within 3 days by macrophages that captured tumor antigens. Within 6 days, naive myeloma-specific CD4+ T cells became activated in draining lymph nodes and subsequently migrated to the incipient tumor site. Upon recognition of tumor-derived antigenic peptides presented on MHC-II by macrophages, the myeloma-specific CD4+ T cells were reactivated and started to secrete cytokines. T cell-derived IFNγ activated macrophages in close proximity to the tumor cells. Tumor cell growth was completely inhibited by such locally activated macrophages. These data indicate a mechanism for immunosurveillance of MHC-II-negative cancer cells by tumor-specific CD4+ T cells through collaboration with macrophages. Gene-targeted mice have recently revealed a role for lymphocytes and interferon-γ (IFNγ) in conferring protection against cancer, but the mechanisms remain unclear. Here, we have characterized a successful primary antitumor immune response initiated by naive CD4+ T cells. Major histocompatibility complex class II (MHC-II)-negative myeloma cells injected subcutaneously into syngeneic mice were surrounded within 3 days by macrophages that captured tumor antigens. Within 6 days, naive myeloma-specific CD4+ T cells became activated in draining lymph nodes and subsequently migrated to the incipient tumor site. Upon recognition of tumor-derived antigenic peptides presented on MHC-II by macrophages, the myeloma-specific CD4+ T cells were reactivated and started to secrete cytokines. T cell-derived IFNγ activated macrophages in close proximity to the tumor cells. Tumor cell growth was completely inhibited by such locally activated macrophages. These data indicate a mechanism for immunosurveillance of MHC-II-negative cancer cells by tumor-specific CD4+ T cells through collaboration with macrophages. The immune system has been proposed to specifically recognize and eliminate newly transformed cells (Burnet, 1970Burnet F.M. The concept of immunological surveillance.Prog. Exp. Tumor Res. 1970; 13: 1-27Crossref PubMed Google Scholar). A series of reports with gene-targeted mice have recently provided strong experimental support for this cancer immunosurveillance hypothesis. Mice deficient for IFNγ, IFNγ receptor, perforin, NKT cells, αβ T cells, γδ T cells, or both T and B cells (Rag2−/−) are all more susceptible to spontaneous or carcinogen-induced cancer (Girardi et al., 2001Girardi M. Oppenheim D.E. Steele C.R. Lewis J.M. Glusac E. Filler R. Hobby P. Sutton B. Tigelaar R.E. Hayday A.C. Regulation of cutaneous malignancy by gammadelta T cells.Science. 2001; 294: 605-609Crossref PubMed Scopus (1) Google Scholar, Kaplan et al., 1998Kaplan D.H. Shankaran V. Dighe A.S. Stockert E. Aguet M. Old L.J. Schreiber R.D. Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice.Proc. Natl. Acad. Sci. USA. 1998; 95: 7556-7561Crossref PubMed Scopus (1094) Google Scholar, Shankaran et al., 2001Shankaran V. Ikeda H. Bruce A.T. White J.M. Swanson P.E. Old L.J. Schreiber R.D. IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity.Nature. 2001; 410: 1107-1111Crossref PubMed Scopus (1895) Google Scholar, Smyth et al., 2000aSmyth M.J. Thia K.Y. Street S.E. Cretney E. Trapani J.A. Taniguchi M. Kawano T. Pelikan S.B. Crowe N.Y. Godfrey D.I. Differential tumor surveillance by natural killer (NK) and NKT cells.J. Exp. Med. 2000; 191: 661-668Crossref PubMed Scopus (641) Google Scholar, Smyth et al., 2000bSmyth M.J. Thia K.Y. Street S.E. MacGregor D. Godfrey D.I. Trapani J.A. Perforin-mediated cytotoxicity is critical for surveillance of spontaneous lymphoma.J. Exp. Med. 2000; 192: 755-760Crossref PubMed Scopus (409) Google Scholar, Street et al., 2002Street S.E. Trapani J.A. MacGregor D. Smyth M.J. Suppression of lymphoma and epithelial malignancies effected by interferon gamma.J. Exp. Med. 2002; 196: 129-134Crossref PubMed Scopus (273) Google Scholar). These studies in animals support earlier observations that humans with a reduced immune capacity are more prone to develop malignancies (Birkeland et al., 1995Birkeland S.A. Storm H.H. Lamm L.U. Barlow L. Blohme I. Forsberg B. Eklund B. Fjeldborg O. Friedberg M. Frodin L. et al.Cancer risk after renal transplantation in the Nordic countries, 1964–1986.Int. J. Cancer. 1995; 60: 183-189Crossref PubMed Scopus (505) Google Scholar, Gatti and Good, 1971Gatti R.A. Good R.A. Occurrence of malignancy in immunodeficiency diseases. A literature review.Cancer. 1971; 28: 89-98Crossref PubMed Scopus (490) Google Scholar). However, the mechanisms of cancer immunosurveillance remain to be elucidated. Our present knowledge of how T cells eliminate cancer is almost exclusively based on memory immune responses investigated with vaccinated mice (Gross, 1943Gross L. Intradermal immunization of C3H mice against a sarcoma that originated in an animal of the same line.Cancer Res. 1943; 3: 326-333Google Scholar, Lynch et al., 1972Lynch R.G. Graff R.J. Sirisinha S. Simms E.S. Eisen H.N. Myeloma proteins as tumor-specific transplantation antigens.Proc. Natl. Acad. Sci. USA. 1972; 69: 1540-1544Crossref PubMed Scopus (297) Google Scholar). Such studies have revealed the critical role of tumor-specific CD4+ T cells in helping cytotoxic CD8+ T cells to kill tumor cells (Ossendorp et al., 1998Ossendorp F. Mengede E. Camps M. Filius R. Melief C.J. Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors.J. Exp. Med. 1998; 187: 693-702Crossref PubMed Scopus (485) Google Scholar). In addition, CD4+ T cells themselves can reject tumors in the absence of CD8+ T cells (Fujiwara et al., 1984Fujiwara H. Fukuzawa M. Yoshioka T. Nakajima H. Hamaoka T. The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2− T cells do not necessarily require recruitment of host’s cytotoxic T cell precursors for implementation of in vivo immunity.J. Immunol. 1984; 133: 1671-1676PubMed Google Scholar, Levitsky et al., 1994Levitsky H.I. Lazenby A. Hayashi R.J. Pardoll D.M. In vivo priming of two distinct antitumor effector populations: the role of MHC class I expression.J. Exp. Med. 1994; 179: 1215-1224Crossref PubMed Scopus (179) Google Scholar, Mumberg et al., 1999Mumberg D. Monach P.A. Wanderling S. Philip M. Toledano A.Y. Schreiber R.D. Schreiber H. CD4(+) T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-gamma.Proc. Natl. Acad. Sci. USA. 1999; 96: 8633-8638Crossref PubMed Scopus (289) Google Scholar). It has been proposed that CD4+ T cells eliminate tumors through activation and recruitment of effector cells, including macrophages and eosinophils (Hung et al., 1998Hung K. Hayashi R. Lafond-Walker A. Lowenstein C. Pardoll D. Levitsky H. The central role of CD4(+) T cells in the antitumor immune response.J. Exp. Med. 1998; 188: 2357-2368Crossref PubMed Scopus (1084) Google Scholar). Several studies suggest that cytokines such as IFNγ that are secreted by type I (Th1) CD4+ T cells might be involved in antitumor and antiangiogenic activities (Mumberg et al., 1999Mumberg D. Monach P.A. Wanderling S. Philip M. Toledano A.Y. Schreiber R.D. Schreiber H. CD4(+) T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-gamma.Proc. Natl. Acad. Sci. USA. 1999; 96: 8633-8638Crossref PubMed Scopus (289) Google Scholar, Qin and Blankenstein, 2000Qin Z. Blankenstein T. CD4+ T cell-mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN gamma receptor expression by nonhematopoietic cells.Immunity. 2000; 12: 677-686Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). Unfortunately, despite these findings in vaccinated mice, it is presently unclear whether similar mechanisms apply in immunosurveillance, i.e., during the course of a primary antitumor immune response. The tumor protective role of CD4+ T cells has been conceptually problematic since most tumor cells do not express MHC-II and thus cannot be directly recognized by tumor-specific CD4+ T cells. Therefore, rejection of MHC-II-negative tumor cells by CD4+ T cells is most likely dependent on professional antigen-presenting cells (APCs) that endocytose, process, and present tumor antigens on their MHC-II to tumor-specific CD4+ T cells. This hypothesis is supported by the observation that dendritic cells isolated from large tumors are loaded with tumor antigens and can activate tumor-specific CD4+ T cells (Dembic et al., 2000Dembic Z. Schenck K. Bogen B. Dendritic cells purified from myeloma are primed with tumor-specific antigen (idiotype) and activate CD4+ T cells.Proc. Natl. Acad. Sci. USA. 2000; 97: 2697-2702Crossref PubMed Scopus (69) Google Scholar, Dembic et al., 2001Dembic Z. Rottingen J.A. Dellacasagrande J. Schenck K. Bogen B. Phagocytic dendritic cells from myelomas activate tumor-specific T cells at a single cell level.Blood. 2001; 97: 2808-2814Crossref PubMed Scopus (28) Google Scholar). However, it is presently not known whether APCs can efficiently activate tumor-specific CD4+ T cells during a primary immune response, when the tumor load is still very low. In order to study the mechanisms of cancer immunosurveillance by CD4+ T cells, we used a T cell receptor (TCR)-transgenic mouse system (Lauritzsen et al., 1994Lauritzsen G.F. Weiss S. Dembic Z. Bogen B. Naive idiotype-specific CD4+ T cells and immunosurveillance of B-cell tumors.Proc. Natl. Acad. Sci. USA. 1994; 91: 5700-5704Crossref PubMed Scopus (124) Google Scholar). In these transgenic mice, T cells recognize a tumor-specific idiotopic (Id) peptide from the secreted immunoglobulin (Ig) L chain V region of the MOPC315 mouse myeloma, presented in the context of MHC-II I-Ed (Bogen et al., 1986Bogen B. Malissen B. Haas W. Idiotope-specific T cell clones that recognize syngeneic immunoglobulin fragments in the context of class II molecules.Eur. J. Immunol. 1986; 16: 1373-1378Crossref PubMed Scopus (172) Google Scholar). The TCR-transgenic mice were made homozygous for the severe combined immunodeficiency (SCID) mutation, which ensures the unique specificity of the T cells by preventing rearrangement of endogenous TCR chains (Bogen et al., 1995Bogen B. Munthe L. Sollien A. Hofgaard P. Omholt H. Dagnaes F. Dembic Z. Lauritzsen G.F. Naive CD4+ T cells confer idiotype-specific tumor resistance in the absence of antibodies.Eur. J. Immunol. 1995; 25: 3079-3086Crossref PubMed Scopus (74) Google Scholar). The high frequency of naive tumor-specific CD4+ T cells in TCR-transgenic mice renders the mice resistant against subcutaneous (s.c.) injection with syngeneic MOPC315 tumor cells, whereas nontransgenic mice develop fatal tumors. Protection is Id specific, CD4+ T cell mediated, and does not require the presence of B cells, γδ T cells, and CD8+ T cells (Bogen et al., 1995Bogen B. Munthe L. Sollien A. Hofgaard P. Omholt H. Dagnaes F. Dembic Z. Lauritzsen G.F. Naive CD4+ T cells confer idiotype-specific tumor resistance in the absence of antibodies.Eur. J. Immunol. 1995; 25: 3079-3086Crossref PubMed Scopus (74) Google Scholar, Lauritzsen et al., 1994Lauritzsen G.F. Weiss S. Dembic Z. Bogen B. Naive idiotype-specific CD4+ T cells and immunosurveillance of B-cell tumors.Proc. Natl. Acad. Sci. USA. 1994; 91: 5700-5704Crossref PubMed Scopus (124) Google Scholar). Importantly, MOPC315 lacks MHC-II and therefore cannot be directly recognized by transgenic Id-specific CD4+ T cells (Dembic et al., 2000Dembic Z. Schenck K. Bogen B. Dendritic cells purified from myeloma are primed with tumor-specific antigen (idiotype) and activate CD4+ T cells.Proc. Natl. Acad. Sci. USA. 2000; 97: 2697-2702Crossref PubMed Scopus (69) Google Scholar, Lauritzsen and Bogen, 1993Lauritzsen G.F. Bogen B. The role of idiotype-specific, CD4+ T cells in tumor resistance against major histocompatibility complex class II molecule negative plasmacytoma cells.Cell. Immunol. 1993; 148: 177-188Crossref PubMed Scopus (76) Google Scholar). However, in large tumors, infiltrating APCs are Id primed and stimulate Id-specific CD4+ T cells (Dembic et al., 2000Dembic Z. Schenck K. Bogen B. Dendritic cells purified from myeloma are primed with tumor-specific antigen (idiotype) and activate CD4+ T cells.Proc. Natl. Acad. Sci. USA. 2000; 97: 2697-2702Crossref PubMed Scopus (69) Google Scholar, Dembic et al., 2001Dembic Z. Rottingen J.A. Dellacasagrande J. Schenck K. Bogen B. Phagocytic dendritic cells from myelomas activate tumor-specific T cells at a single cell level.Blood. 2001; 97: 2808-2814Crossref PubMed Scopus (28) Google Scholar). Rejection of MOPC315 by the Id-specific TCR-transgenic mice does not require immunization of the mice, and thus represents a genuine primary immune response. It has been difficult to study the mechanisms of tumor rejection in Id-specific TCR-transgenic mice, because the myeloma cells could not be precisely localized in vivo after injection. To solve this, we have in this study embedded injected tumor cells in a collagen gel (Matrigel), which enabled us to analyze the early interactions between tumor cells and infiltrating cells from the host, during a primary antitumor immune response. In accordance with a previous report (Bogen et al., 1995Bogen B. Munthe L. Sollien A. Hofgaard P. Omholt H. Dagnaes F. Dembic Z. Lauritzsen G.F. Naive CD4+ T cells confer idiotype-specific tumor resistance in the absence of antibodies.Eur. J. Immunol. 1995; 25: 3079-3086Crossref PubMed Scopus (74) Google Scholar), Id-specific TCR-transgenic SCID mice were resistant against s.c. challenge with MOPC315 cells injected in phosphate-buffered saline (PBS), whereas nontransgenic SCID littermates developed fatal tumors (Figure 1A ). To estimate the kinetics of rejection, serum concentrations of myeloma protein M315 were measured at several time points after injection by enzyme-linked immunosorbent assay (ELISA) (Figure 1B). In most control SCID mice, M315 levels increased exponentially, reflecting progressive s.c. tumor growth (Figure 1B, left). By contrast, in all TCR-transgenic SCID mice, M315 levels were low (<350 ng/ml) throughout the experiment (Figure 1B, right). In 9 out of these 18 TCR-transgenic SCID mice, M315 remained undetectable in serum (detection level: 1 ng/ml). In the other 9 mice, the serum concentration of M315 increased slowly until days 8–11, suggesting that the tumor cells underwent a limited in vivo expansion during the first days after injection. After day 11, however, serum M315 levels decreased in all TCR-transgenic SCID mice, reflecting the elimination of the tumor cells. These data strongly suggest that the effector functions of the antitumor immune response are activated well before day 11 after injection, and that the tumor cells are completely rejected by day 15. In order to precisely localize the injected tumor cells in the host, we embedded MOPC315 cells in a collagen gel (Matrigel) that is soluble at +4°C but gels at body temperature, resulting in a plug that can easily be identified in vivo (Kleinman et al., 1986Kleinman H.K. McGarvey M.L. Hassell J.R. Star V.L. Cannon F.B. Laurie G.W. Martin G.R. Basement membrane complexes with biological activity.Biochemistry. 1986; 25: 312-318Crossref PubMed Scopus (1173) Google Scholar). Another advantage of this technique is that it traps the infiltrating host cells (see below). TCR-transgenic SCID mice were effectively protected against MOPC315 injected in Matrigel for more than 50 days after injection (Figure 1C). However, most transgenic mice failed to completely reject the myeloma cells injected in Matrigel, as revealed by low but sustained levels of serum M315 (Figure 1D). As a consequence of this incomplete rejection, slow-growing tumors developed in three out of seven TCR-transgenic SCID mice as late as 60–90 days after the injection (Figure 1C). As with MOPC315 injected in PBS, the antitumor immune response against MOPC315 in Matrigel was rapid, since as early as 12 days after injection, serum M315 levels were significantly lower in transgenic mice compared to SCID controls (Figure 1D, right). Proliferation of tumor-specific CD4+ T cells was first observed in the lymph node (LN) draining the injection site at day 3 and dramatically increased at day 6 after injection of MOPC315 in either Matrigel or PBS (Figure 2A and data not shown). This clonal expansion was associated with upregulation of the activation marker CD69 on most tumor-specific CD4+ T cells (Figure 2A). The immune response was local, as similar changes were not observed in nondraining LN or spleen (data not shown; also, see below). The T cell activation was not due to Matrigel per se because injection of cell-free Matrigel gave no response on day 6. Moreover, T cell activation was Id specific since no response was seen after injection with the control J558 myeloma, which secretes a monoclonal IgA with V regions different from those of MOPC315 (Figure 2A). This is in accordance with previous reports showing that the TCR-transgenic mice reject MOPC315, but not J558 cells (Bogen et al., 1995Bogen B. Munthe L. Sollien A. Hofgaard P. Omholt H. Dagnaes F. Dembic Z. Lauritzsen G.F. Naive CD4+ T cells confer idiotype-specific tumor resistance in the absence of antibodies.Eur. J. Immunol. 1995; 25: 3079-3086Crossref PubMed Scopus (74) Google Scholar, Lauritzsen et al., 1994Lauritzsen G.F. Weiss S. Dembic Z. Bogen B. Naive idiotype-specific CD4+ T cells and immunosurveillance of B-cell tumors.Proc. Natl. Acad. Sci. USA. 1994; 91: 5700-5704Crossref PubMed Scopus (124) Google Scholar). From days 3–9 after tumor cell injection, the tumor-specific CD4+ T cells differentiated in draining LN from naive to memory phenotype: the cells increased in size (blast formation), upregulated surface CD11a and CD44 molecules, downregulated CD62L, and synthesized DNA (i.e., incorporated bromodeoxyuridine, BrdU) (Figure 2C, left and data not shown). Since CD44hiCD62Llo T cells have the capacity to enter nonlymphoid tissues, these data prompted us to analyze the cellular content of MOPC315-containing Matrigel plugs. At day +6, a small but distinct population of Matrigel-infiltrating CD69+ tumor-specific T cells could be detected (Figure 2B). At day +9, Matrigel-infiltrating tumor-specific T cells were more frequent and had a typical memory phenotype (enlarged size, CD11ahi, CD44hi, CD62Llo/−, BrdU+) (Figure 2C, right). Importantly, these Matrigel-infiltrating tumor-specific T cells were producing the Th1 cytokines IFNγ and tumor necrosis factor α (TNFα) at day +11 (Figure 2D). In the same experiment, interleukin-2, but not granulocyte/macrophage colony-stimulating factor (GM-CSF), was detected in Matrigel-infiltrating tumor-specific CD4+ T cells (data not shown). These results demonstrate that 11 days are sufficient for the priming of tumor-specific CD4+ T cells in draining LN, the migration of primed T cells into the tissue in which the tumor cells are located, and the secretion of cytokines. The early in vivo interactions between the tumor cells and cells of the immune system were visualized by immunostaining of MOPC315-containing Matrigel plugs in TCR-transgenic SCID mice (Figure 3). Figure 3A shows a s.c. day +6 Matrigel plug containing islets of tumor cells (arrows). Recruitment of blood leukocytes toward the MOPC315-containing Matrigel plug was suggested by the appearance of nucleated cells within blood vessels at the periphery of the plug (day +3, Figures 3B and 3C). At day +6, the nucleated (Hoechst+) cells inside the Matrigel plug can basically be divided into two distinct populations: (i) the MOPC315 cells growing in islets and stained by the myeloma marker CD138 (syndecan-1), and (ii) the host cells that essentially all expressed MHC-II (Figure 3D). These MHC-II+ host cells formed a dense layer covering the edge of the myeloma-containing Matrigel plug 3–6 days after injection (arrows in Figures 3D and 3F). Numerous MHC-II+ host cells were seen penetrating the Matrigel plug, of which several were in close contact with the CD138+ myeloma cells (arrows in Figure 3E). The Matrigel-infiltrating MHC-II+ cells coexpressed the macrophage marker F4/80 (day +3, Figure 3F ). Figure 3G shows a MHC-II+ cell crossing the endothelium of a blood vessel inside a Matrigel plug. Collectively, these data suggest that there is a massive recruitment of host MHC-II+ macrophages toward the tumor cells. These macrophages are most likely derived from blood monocytes that extravasated mainly from vessels situated at the periphery of the plug, but also from vessels surrounded by the gel. Additionally, infiltrating T cells could be detected in Matrigel sections, but they were much fewer than the macrophages. Importantly, some tumor-specific T cells apparently made contact with MHC-II+ macrophages in the MOPC315-containing Matrigel plug (Figure 3H).Figure 4Matrigel-Infiltrating Macrophages Become Activated in TCR-Transgenic MiceShow full caption(A) A TCR-transgenic SCID mouse was injected with GFP-labeled MOPC315 cells in Matrigel. The cellular content of the Matrigel plug was analyzed by flow cytometry at day +5, revealing three distinct populations: CD11b+ cells (R1), GFP−CD11b− cells (R2), and GFP+ tumors cells (R3). MHC-II expression was almost exclusively restricted to the CD11b+ population (R1), while the myeloma cells (R3) were negative for MHC-II.(B) TCR-transgenic SCID or SCID mice were injected with Matrigel containing MOPC315. The expression of various markers on gated Matrigel-infiltrating CD11b+ cells was analyzed by flow cytometry at day +6.(C) MOPC315 cells were injected s.c. in Matrigel, and MHC-II expression on Matrigel-infiltrating CD11b+ cells was measured by flow cytometry at day +5 in a TCR-transgenic SCID mouse (shaded area) as compared to a SCID mouse (boldface line).(D) TCR-transgenic SCID or SCID mice were injected with either MOPC315-containing Matrigel or cell-free, PBS-containing Matrigel. At various time points after injection, the Matrigel-infiltrating CD11b+ cells were counted and divided into MHC-II high (hatched area) or low (blank area) expressers, as defined in (C). Each column represents the mean of three mice (pooled organs). T cell activation in draining LN was analyzed in parallel and the data are represented in Figure 2A. Dotted lines in (A), (B), and (C) indicate an isotype-matched control mAb. TG, transgenic.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) A TCR-transgenic SCID mouse was injected with GFP-labeled MOPC315 cells in Matrigel. The cellular content of the Matrigel plug was analyzed by flow cytometry at day +5, revealing three distinct populations: CD11b+ cells (R1), GFP−CD11b− cells (R2), and GFP+ tumors cells (R3). MHC-II expression was almost exclusively restricted to the CD11b+ population (R1), while the myeloma cells (R3) were negative for MHC-II. (B) TCR-transgenic SCID or SCID mice were injected with Matrigel containing MOPC315. The expression of various markers on gated Matrigel-infiltrating CD11b+ cells was analyzed by flow cytometry at day +6. (C) MOPC315 cells were injected s.c. in Matrigel, and MHC-II expression on Matrigel-infiltrating CD11b+ cells was measured by flow cytometry at day +5 in a TCR-transgenic SCID mouse (shaded area) as compared to a SCID mouse (boldface line). (D) TCR-transgenic SCID or SCID mice were injected with either MOPC315-containing Matrigel or cell-free, PBS-containing Matrigel. At various time points after injection, the Matrigel-infiltrating CD11b+ cells were counted and divided into MHC-II high (hatched area) or low (blank area) expressers, as defined in (C). Each column represents the mean of three mice (pooled organs). T cell activation in draining LN was analyzed in parallel and the data are represented in Figure 2A. Dotted lines in (A), (B), and (C) indicate an isotype-matched control mAb. TG, transgenic. We next used flow cytometry to characterize the Matrigel-infiltrating MHC-II+ cells 1–6 days after injection. For these experiments, green fluorescent protein (GFP)-transduced MOPC315 cells were used, allowing a simple and effective detection of tumor cells. Analysis of the cellular content of a Matrigel plug at day +5 revealed a massive infiltration of cells expressing the CD11b (Mac-1) myeloid cell marker (Figure 4A). MHC-II expression was almost exclusively restricted to these CD11b+ cells (R1 in Figure 4A), whereas GFP+ MOPC315 cells were MHC-II negative (R3). A large population of CD11b−GFP− cells that were essentially devoid of MHC-II expression was also detected in Matrigel (Figure 4A, R2). Most of these triple-negative cells were most likely MOPC315 cells that had lost GFP expression (unpublished data), compatible with the immunostaining data which show that essentially all MHC-II-negative cells in Matrigel stain positively for the myeloma marker CD138 (Figure 3D). Further characterization of the Matrigel-infiltrating CD11b+ cells identified the cells as typical macrophages: they expressed CD11a (LFA-1 α chain), CD54 (ICAM-1), CD80, CD86 and Mac-3 (Figure 4B), while they were negative for CD45R/B220, CD4 and CD8 (not shown). Interestingly, a minority (4%–12%) of the Matrigel-infiltrating CD11b+ cells expressed CD11c+, indicating a small subset of dendritic cells (Figure 4B). The immunostaining data revealed that some tumor-specific CD4+ T cells made close contact with macrophages inside MOPC315-containing Matrigel plugs (Figure 3H). A functional consequence of such an interaction could be the activation of the macrophages by the CD4+ T cells. Indeed, after MOPC315 injections, levels of the activation marker MHC-II on Matrigel-infiltrating macrophages were dramatically increased in TCR-transgenic SCID mice as compared to nontransgenic SCID mice (Figure 4C). Moreover, two additional macrophage activation markers (CD11a and CD54) were upregulated on infiltrating macrophages in TCR-transgenic SCID mice (Figure 4B). This prompted us to investigate the kinetics of macrophage recruitment and activation. Large numbers of macrophages were found in Matrigel plugs containing MOPC315 as early as 3 days after injection, but, at this time point, MHC-II expression was not upregulated (Figure 4D). In contrast, at day +6, most Matrigel-infiltrating CD11b+ cells had upregulated MHC-II in TCR-transgenic mice (Figure 4D), correlating with the influx of tumor-specific CD4+ T cells at the same time point (Figure 2B). Interestingly, the recruitment of macrophages was dependent on the presence of myeloma cells and was not caused by the Matrigel itself since very few macrophages infiltrated cell-free Matrigel plugs (Figure 4D). In order to demonstrate that the observed macrophage activation was mediated by the tumor-specific CD4+ T cells, we performed two in vivo experiments with blocking monoclonal antibodies (mAb). First, we used an anti-CD4 mAb to deplete CD4+ T cells in TCR-transgenic SCID mice. The activation of Matrigel-infiltrating macrophages was completely blocked in such CD4+ T cell-depleted mice (Figure 5A ). In a second experiment, we took advantage of an anti-MHC-II I-E mAb (Dembic et al., 2004Dembic Z. Hofgaard P.O. Omholt H. Bogen B. Anti-class II antibodies, but not cytotoxic T-lymphocyte antigen 4-immunoglobulin hybrid molecules, prevent rejection of major histocompatibility complex class II-negative myeloma in T-cell receptor-transgenic mice.Scand. J. Immunol. 2004; 60: 143-152Crossref PubMed Scopus (3) Google Scholar) to block the activation of tumor-specific CD4+ T cells in draining LN (Figure 5B). Such blocking of T cell activation inhibited the migration of T cells into Matrigel (Figure 5C), indicating that priming in draining LN is a prerequisite for the migration of tumor-specific CD4+ T cells to the incipient tumor site. Moreover, blocking of T cell activation and migration with anti-MHC-II mAb completely inhibited macrophage activation in the Matrigel plugs, as measured by MHC-II I-A and CD11a levels (Figures 5D and 5E). The antigen specificity of macrophage activation by CD4+ T cells was tested by injecting Id-specific TCR-transgenic SCID mice with either MOPC315 or the control J558 myeloma (Figure 6A ). In contrast to MOPC315, injections with J558 did not result in macrophage activation in TCR-transgenic SCID mice (Figure 6A). In the same experiment, nontransgenic SCID mice were injected with MOPC315 and J558, and this confirmed that CD4+ T cells are needed for macrophage activation in MOPC315-containing Matrigel (Figure 6A). These results suggest that macrophage activation is the result of CD4+ T cell recognition of tumor antigens presented on MHC-II molecules by Matrigel-infiltrating macrophages. We therefore examined whether Matrigel-infiltrating macrophages could present tumor-derived Id peptides. Macrophages that had infiltrated MOPC315-containing Matrigel plugs were purified and tested for their ability to spontaneously stimulate Id-specific CD4+ T cells in vitro. A modest proliferation of tumor-specific CD4+ T cells could be detected in the absence of added peptide, indicating an in vivo loading of the macrophages (Figure 6B). Addition of exogenous synthetic Id peptide to the cultures further improved the proliferation, demonstrating that the Matrigel-infiltrating macrophages were potent APCs (Figure 6B)." @default.
• W2050161362 created "2016-06-24" @default.
• W2050161362 creator A5025472514 @default.
• W2050161362 creator A5026062890 @default.
• W2050161362 creator A5036473031 @default.
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• W2050161362 date "2005-03-01" @default.
• W2050161362 modified "2023-10-18" @default.
• W2050161362 title "Primary Antitumor Immune Response Mediated by CD4+ T Cells" @default.
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